The chaperone heat shock protein 90 (Hsp90) is an emerging target in cancer treatment due to its important roles in regulating key proteins in cell growth, survival, and differentiation pathways. Hsp90 inhibitors may have further medical use in the treatment of viral infections and inflammatory conditions. Hsp90 assists the folding, maturation, stability, and trafficking of a specific group of proteins called client proteins.
Hsp90 function is regulated by a pocket in the N-terminal region of the protein that binds and hydrolyzes ATP. Occupancy of this pocket by high affinity ligands prevents the Hsp90 client proteins from achieving their mature functional conformation. Protein clients of Hsp90 are mostly kinases, steroid receptors, and transcriptional factors involved in driving multistep malignancy and, in addition, mutated oncogenic proteins required for the transformed phenotype. Examples include Her2, Raf-1, Akt, Cdk4, cMet, mutant p53, ER, AR, mutant BRaf, Bcr-Abl, Flt-3, Polo-1 kinase, HIF-1 alpha, and hTERT (see Therapeutic and diagnostic implications of Hsp90 activation. Trends Mol. Med. 2004, 10, 283-290; Hsp90 inhibitors as novel cancer chemotherapeutic agents. Trends Mol. Med. 2002, 8, S55-S61; and Hsp90 as a new therapeutic target for cancer therapy: the story unfolds. Expert Opin. Biol. Ther. 2002, 2, 3-24).
The past few years have witnessed a tremendous growth in the discovery of Hsp90-specific inhibitors belonging to several distinct chemical classes, which include benzoquinone ansamycins (e.g. geldanamycin derivatives), radicicol derivatives, purine-scaffold-based inhibitors, dihydroxyphenylpyrazoles, and small peptides. Among them, 17-AAG and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), derivatives of the natural product geldanamycin, are currently under evaluation in multiple clinical trials. 17-AAG inhibits Hsp90 by binding competitively to its N-terminal ATP binding site. This site is highly conserved among Hsp90 family proteins, whose human members include cytoplasmic Hsp90α and Hsp90β, ER-resident Grp94, and mitochondrial tumour necrosis factor receptor-associated protein 1 (Trap1). The Hsp90 chaperone complex facilitates the folding of client proteins through coupled cycles of ATP hydrolysis. Thus, inhibition of the ATPase activity results in misfolding and degradation of client proteins via the ubiquitin-proteasome pathway and in turn leads to growth arrest or apoptosis in cancer cells (see J. Med. Chem. 2006, 49, 4606-4615).
Hsp90 is over expressed (about 2-20 fold) in multiple tumour types as a result of oncogenic transformation (e.g. accumulation of mutated proteins) and cellular stress (e.g. low pH and lack of nutrients). Cancer cells are very adaptive to hostile microenvironments and are capable of acquiring drug resistance, in part due to their inherent genetic instability and plasticity. Hence, a need exists for inhibitors of Hsp90 to combat a variety of hard-to-treat tumours by disrupting concurrently a wide range of oncogenic pathways.
More recently it became apparent that Hsp90 function is also required to sustain viral infections caused for instance by vesicular stomatitis virus, paramyxovirus SV5, HPIV-2, HPIV-3, SV41 and LaCrosse bunyavirus (Antiviral activity and RNA polymerase degradation following Hsp90 inhibition in a range of negative strand viruses. Virology 2007, Epub ahead of print). The Hsp90 inhibitors geldanamycin and radicicol were previously shown to block the replication of human cytomegalovirus (HCMV) and herpes simplex virus type 1 in relevant cell culture systems (Geldanamycin, a potent and specific inhibitor of Hsp90, inhibits gene expression and replication of human cytomegalovirus. Antivir. Chem. Chemother. 2005, 16, 135-146; Geldanamycin, a ligand of heat shock protein 90, inhibits the replication of herpes simplex virus type 1 in vitro. Antimicrob. Agents Chemother. 2004, 48, 867-872). Geldanamycin and radicicol treated cells fail to mount a NFkappaB-dependent anti-viral response and Hsp90 function has been shown to be required for proper NFkappaB signalling (Requirement of Hsp90 activity for IkappaB kinase (IKK) biosynthesis and for constitutive and inducible IKK and NFkappaB activation. Oncogene 2004, 23, 5378-5386). The NFkappaB signalling pathway is likewise operative in inflammatory conditions and the reported anti-inflammatory activity of geldanamycin is potentially explained by the failure of function as a transcription factor of NFkappaB in absence of Hsp90 chaperone function (Disruption of Hsp90 function results in degradation of the death domain kinase, receptor-interacting protein (RIP), and blockage of tumor-necrosis factor induced nuclear factor-κB activation. J. Biol. Chem. 2000, 275, 10519-10526; Geldanamycin inhibits NFkappaB activation and interleukin-8 gene expression in cultured human respiratory epithelium. Am. J. Respir. Cell Mol. Biol. 2001, 25, 92-97). Alternatively the anti-inflammatory function of the Hsp90 inhibitor may be related to the fact that the glucocorticoid receptor is a client protein for Hsp90 that does not function properly as a transcriptional regulator of pro- and anti-inflammatory genes in absence of Hsp90 function (Isolation of Hsp90 mutants by screening for decreased steroid receptor function. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 11424-11428; Geldanamycin, an inhibitor of heat shock protein 90 (Hsp90) mediated signal transduction has anti-inflammatory effects and interacts with glucocorticoid receptor in vivo. Br. J. Pharmacol. 2000, 131, 13-16).
In summary, due to its pleiotropic effect on central regulatory molecules like kinases, transcription factors and hormone receptors, novel Hsp90 inhibitors may have medical utility not only in cancer but also for the treatment of viral infections and inflammatory disease states like rheumatoid arthritis, Crohn's disease, etc.
WO 2006/113498 and WO 2007/041362 relate respectively to 2-aminoquinazolin-5-one and 2-amino-7,8-dihydro-6H-pyrido[4,3-d]pyrimidin-5-one compounds which are Hsp90 inhibitors. The present inventors have devised alternative Hsp90 inhibitors and have, surprisingly, found that the ketone group in these prior art compounds can be replaced by a group with increased bulk and functionality without loss of activity and, in some cases, with increased activity.
Therefore, in a first aspect of the present invention, there is provided a compound of general formula (I):
or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein:
R1 is selected from hydrogen, halogen, hydroxyl, amino, thiol, C1-C6 alkoxy, C1-C6 alkylthiol, C1-C10 alkyl, C1-C6 alkylamino, arylamino, aryl(C1-6 alkyl)amino, aryl, heteroaryl, C3-C7 cycloalkyl, or C3-C7 heterocyclyl, any of which may optionally be substituted;
R2 and R3 are each independently hydrogen, halogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C7 cycloalkyl, C5-C7 cycloalkenyl, aryl, heteroaryl, or C3-C7 heterocyclyl, any of which may optionally be substituted; R2 and R3 may also form a 3 to 6 membered Spiro ring system, optionally fused with an aryl or heteroaryl ring;
R4, R5, R8 and R9 are each independently selected from hydrogen, halogen, C1-C6 alkyl, —OR7, —SR7, —NR7R7′, —OC(O)R7′, —N(R7)C(O)R7′, or —N(R7)SO2R7′; R4 and R9 and/or R5 and R8 may also form a 3 to 6 membered Spiro ring system, optionally fused with an aryl or heteroaryl ring;                R7 and R7′ are each independently hydrogen, C1-C6 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, C3-C7 cycloalkyl, C5-C7 cycloalkenyl, aryl, heteroaryl, or C3-C7 heterocyclyl, any of which may optionally be substituted;        or,        when R4, R5, R8 or R9 is —OC(O)R7′, —N(R7)C(O)R7′, or —N(R7)SO2R7′, R7′ may additionally be NR10R11, where R10 and R11 are each independently hydrogen or C1-C6 alkyl;        and        
R6 is hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, (CH2)nC(O)R12, C1-C6 alkylN(R14)2, C3-C7 cycloalkyl, C5-C7 cycloalkenyl, aryl, heteroaryl, or C3-C7 heterocyclyl, any of which may optionally be substituted;
n is 0 to 4;
R12 is C1-C6 alkyl, OH, O(C1-C6 alkyl) or N(R13)2;
where:                each R13 is independently hydrogen, methyl or ethyl, or the two R13 groups together with the nitrogen atom to which they are attached form a 5 to 7 membered heterocyclic ring optionally substituted and optionally containing a further hetero atom, selected from N optionally substituted, O or S;        each R14 is independently hydrogen, C1-C6 alkyl, or the two R14 groups together with the nitrogen atom to which they are attached form a 5 to 7 membered heterocyclic ring optionally substituted and optionally containing a further hetero atom, selected from N optionally substituted, O or S.The compounds of the invention have Hsp90 inhibitory activity and are therefore useful for the treatment of diseases and conditions which are mediated by excessive or inappropriate Hsp90 activity such as cancers, viral infection and inflammatory diseases or conditions.        
In the context of the present specification, the term “C1-C6 alkyl” refers to a fully saturated straight or branched saturated hydrocarbon chain having one to six carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, t-butyl, n-hexyl, methylenecyclopropyl, methylenecyclobutyl and methylenecyclopentyl. “C1-C3 alkyl” and “C1-C10 alkyl” have similar meanings except that they contain from one to three and from one to ten carbon atoms respectively.
The term “C2-C10 alkenyl” refers to a straight or branched hydrocarbon chain having from two to ten carbon atoms and at least one carbon-carbon double bond. Examples include ethenyl, 2-propenyl and isobutenyl. “C2-C5 alkenyl” and “C2-C6 alkenyl” have similar meanings except that they contain from two to five and from two to six carbon atoms respectively.
The term “C2-C10 alkynyl” refers to a straight or branched hydrocarbon chain having from two to ten carbon atoms and at least one carbon-carbon triple bond. Examples include ethynyl, 2-propynyl and isobutynyl. “C2-C5 alkynyl” and “C2-C6 alkynyl” have similar meanings except that they contain from two to five and from two to six carbon atoms respectively.
When alkyl, alkenyl and alkynyl groups are substituted, suitable substituents include one or more halo, OH, SH, O(C1-C6 alkyl), S(C1-C6 alkyl), nitro, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C7 cycloalkyl, C3-C7 heterocyclyl, aryl, heteroaryl, —O(C3-C7 cycloalkyl), —O(C3-C7 heterocyclyl), —O(aryl) or —O(heteroaryl) groups.
“C3-C7 cycloalkyl” refers to a saturated 3 to 7 membered carbocyclic ring. Examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. “C3-C11 cycloalkyl” has similar meanings except that it contains from 3 to 11 membered carbocyclic ring.
“C5-C7 cycloalkenyl” refers to a 5 to 7 membered carbocyclic ring having at least one ring carbon-carbon double bond. “C5-C11 cycloalkenyl” has similar meanings except that it contains from 5 to 11 membered carbocyclic ring.
“C3-C7 heterocyclyl” refers to a 3 to 7 membered ring system having at least one heteroatom chosen from N, O or S and optionally being partially unsaturated. Examples of such groups include morpholino, pyrrolidino, piperidinyl, piperazinyl, tetrahydrofuranyl. “C3-C11 heterocyclyl” has similar meanings except that it contains from 3 to 11 membered ring system.
In the present specification, “halo” or “halogen” refer to fluoro, chloro, bromo or iodo.
The term “aryl” in the context of the present specification refers to a ring system having from 5 to 14 ring carbon atoms and containing up to three rings, at least one of which has aromatic character. Examples of aryl groups are benzene, biphenyl and naphthalene.
The term “heteroaryl” in the context of the present specification refers to a ring system having from 5 to 14 ring atoms, one or more of which is a heteroatom selected from N, O and S and containing up to three rings, at least one of which has aromatic character. Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, oxazolyl, furanyl, thienyl, quinolinyl, isoquinolyl, quinazolyl, thiazolyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, indolyl, indazolyl, imidazolyl, benzimidazolinyl and benzodioxolyl ring systems.
When cycloalkyl, heterocyclyl, aryl and heteroaryl groups are substituted, there may be one or more substituents selected from:
(i) C1-C10 alkyl, C2-C10 alkenyl or C2-C10 alkynyl, any of which may be substituted as defined above; or
(ii) C3-C7 cycloalkyl, C5-C7 cycloalkenyl, aryl, heteroaryl, C3-C7 heterocyclyl, any of which may, in turn, be substituted with one or more substituents selected from halogen, —OR10, —SR10, —NR10R10′, —C(O)R10, —CO2R10, —C(O)NR10R10′, —S(O)R10, —SO2R10, —SO2NR10R10′, —OC(O)R10′, —N(R10)C(O)R10′, —N(R10)SO2R10′, —CN, or —NO2; wherein R10 and R10′ are each independently hydrogen, C1-C6 alkyl, C2-C5 alkenyl or C2-C5alkynyl; or
(iii) —OR11, —SR11, —NR11R11′, —C(O)R11, —CO2R11, —C(O)NR11R11′, —S(O)R11, —SO2R11, or —SO2NR11R11′, —OC(O)R11′, —N(R11)C(O)R11′, or —N(R11)SO2R11′, halogen, —CN, or —NO2; wherein R11 and R11′ are each independently hydrogen, C1-C6 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, C3-C11 cycloalkyl, C5-C11 cycloalkenyl, aryl, heteroaryl, or C3-C11 heterocyclyl.
The term “C1-C6 alkoxy” refers to the group C1-C6 alkyl-O—.
The term “C1-C6 alkylthiol” refers to the group C1-C6 alkyl-S—.
The term “C1-C6 alkylamino” refers to the group C1-C6 alkyl attached to an amino moiety.
Appropriate pharmaceutically and veterinarily acceptable salts of the compounds of general formulae (I) and (II) include basic addition salts such as sodium, potassium, calcium, aluminium, zinc, magnesium and other metal salts as well as choline, diethanolamine, ethanolamine, ethyl diamine and other well known basic addition salts.
Where appropriate, pharmaceutically or veterinarily acceptable salts may also include salts of organic acids, especially carboxylic acids, including but not limited to acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, pamoate, pectinate, 3-phenylpropionate, picrate, pivalate, proprionate, tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate; organic sulfonic acids such as methanesulfonate, ethanesulfonate, 2-hydroxyethane sulfonate, camphorsulfonate, 2-naphthalenesulfonate, benzenesulfonate, p-chlorobenzenesulfonate and p-toluenesulfonate; and inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, hemisulfate, thiocyanate, persulfate, phosphoric and sulfonic acids.
Salts which are not pharmaceutically or veterinarily acceptable may still be valuable as intermediates.
Prodrugs are any covalently bonded compounds which release the active parent drug according to general formula (I) in vivo.
In the compounds of the first aspect of the invention, it is greatly preferred that, independently or in any combination:
R8 is H; and
R9 is H.
Other preferred compounds include those in which R1 is hydrogen or C1-C6 alkyl, which may optionally be substituted with halo. It is more preferred that R1 is hydrogen or C1-C3 alkyl, but particularly useful compounds are those in which R1 is hydrogen, methyl or ethyl.
More active compounds of general formula (I) include those in which, in addition to R8 and R9, R4 and R5 are also hydrogen.
In other preferred compounds of general formula (I), one or both of R2 and R3 is aryl, heteroaryl, C3-C7 cycloalkyl, C3-C7 heterocyclyl or C1-C6 alkyl, any of which may optionally be substituted with one or more substituents chosen from halogen, OH, C1-C6 alkoxy, O—C3-C7 cycloalkyl, aryl, heteroaryl, C3-C7 heterocyclyl, O—C3-C7 heterocyclyl, O-aryl, O-heteroaryl moieties, or, except when R2 or R3 is alkyl, C1-C6 alkyl, any of which may be substituted with methyl or halo.
It is more preferred that one of R2 and R3 is as defined above and that the other of R2 and R3 is hydrogen.
Particularly useful compounds include those in which R2 is hydrogen and R3 is furanyl, thienyl, phenyl or benzo[1,3]dioxolyl, any of which may be substituted by one or more halo, methyl, methoxy, hydroxyl or phenyl, pyridyl, pyrazole, indolyl, methylpyrazole, morpholino groups, any of which may optionally be substituted
Especially preferred R3 groups include 2-methoxyphenyl, 2-fluorophenyl, 2-bromophenyl, 2-bromo-4-fluorophenyl, 4-methylphenyl, 3-fluorophenyl, 4-fluorophenyl, 4-chlorophenyl, phenyl, 2,6-dimethoxyphenyl, 2,6-difluorophenyl, 2,4-difluorophenyl, 2-morpholinophenyl, 1-(2-phenoxyethanol), 4-benzo[1,3]dioxolyl, biphenyl, pyridylphenyl, for example 2-pyridylphenyl such as 2-(2-pyridyl)phenyl, 2-(3-pyridyl)phenyl and 2-(4-pyridyl)phenyl, 4-fluoro-2-pyridylphenyl, indolylphenyl, for example 2-(1H-indol-7-yl)-phenyl, 2-(1H-indol-4-yl)-phenyl, 2(1-methylpyrazol-4-yl)phenyl and 4-fluoro-2(1-methylpyrazol-4-yl)phenyl.
In other preferred compounds of general formula (I), R6 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted aryl; or R6 is C(O)C1-C6 alkyl, (CH2)nC(O)OH, (CH2)nC(O)O(C1-C2 alkyl), (CH2)nC(O)-morpholino or C1-C6 alkylN(R14)2, where R14 and n are as defined above.
In particular, R6 is hydrogen, methyl, ethyl, propyl, butyl, hexynyl, phenyl, —C1-C3 alkylN(C1-C2 alkyl)2, morpholino(C1-C3 piperazinyl(C1-C3 alkyl)-, 4-methylpiperazinyl(C1-C3 pyrrolidino(C1-C3 alkyl), —C(O)methyl, —(CH2)1-3C(O)OH, —(CH2)1-3C(O)O(C1-C2 alkyl) or —CH2C(O)-morpholino.
In one embodiment of the invention, the compound 2-amino-7,7-dimethyl-7,8-dihydro-6H-quinazolin-5-one oxime (i.e. a compound of formula (I) in which R1, R4, R5, R6, R8 and R9 are all hydrogen and R2 and R3 are both methyl) is specifically excluded from the scope of the compounds of the invention.
In a preferred embodiment, the compounds of general formula (I) have an IC50 value for inhibiting Hsp90 activity less than or equal to 100 μM. In more preferred embodiments, the IC50 value is less than or equal to 50 μM, even more preferred with an IC50 value less than or equal to 25 μM. Still more preferred embodiment have IC50 values less than or equal to 10 μM, and even more preferred embodiments have IC50 values less than or equal to 1 μM, A representative assay for determining Hsp90 inhibitory activity is described in Example 7.
Particularly preferred compounds of general formula (I) include:    1. 2-Amino-7-(4-chloro-phenyl)-7,8-dihydro-6H-quinazolin-5-one oxime    2. 2-Amino-7-(4-chloro-phenyl)-7,8-dihydro-6H-quinazolin-5-one O-methyl-oxime    3. 2-Amino-7-phenyl-7,8-dihydro-6H-quinazolin-5-one oxime    4. 2-Amino-7-phenyl-7,8-dihydro-6H-quinazolin-5-one O-methyl-oxime    5. 2-Amino-7-phenyl-7,8-dihydro-6H-quinazolin-5-one oxime-O-acetyl    6. 2-Amino-7-(4-fluoro-phenyl)-7,8-dihydro-6H-quinazolin-5-one oxime    7. 2-Amino-7-(4-fluoro-phenyl)-7,8-dihydro-6H-quinazolin-5-one O-methyl-oxime    8. 2-Amino-7-(4-fluoro-phenyl)-7,8-dihydro-6H-quinazolin-5-one O-ethyl-oxime    9. [2-Amino-7-(4-fluoro-phenyl)-7,8-dihydro-6H-quinazolin-5-ylideneaminooxy]-acetic acid    10. 2-Amino-7-(4-fluoro-phenyl)-7,8-dihydro-6H-quinazolin-5-one O-(2-morpholin-4-yl-2-oxo-ethyl)-oxime    11. 2-Amino-7-(4-fluoro-phenyl)-7,8-dihydro-6H-quinazolin-5-one O-propyl-oxime    12. 2-Amino-7-(4-fluoro-phenyl)-7,8-dihydro-6H-quinazolin-5-one O-butyl-oxime    13. 4-[2-Amino-7-(4-fluoro-phenyl)-7,8-dihydro-6H-quinazolin-5-ylideneaminooxy]-butyric acid ethyl ester    14. 4-[2-Amino-7-(4-fluoro-phenyl)-7,8-dihydro-6H-quinazolin-5-ylideneaminooxy]-butyric acid    15. 2-Amino-7-(4-fluoro-phenyl)-7,8-dihydro-6H-quinazolin-5-one O-(2-morpholin-4-yl-ethyl)-oxime    16. 2-Amino-7-(2-methoxy-phenyl)-7,8-dihydro-6H-quinazolin-5-one oxime    17. 2-Amino-7-(2-methoxy-phenyl)-7,8-dihydro-6H-quinazolin-5-one O-methyl-oxime    18. 2-Amino-7-thien-2-yl-7,8-dihydro-6H-quinazolin-5-one oxime    19. 2-Amino-7-thien-2-yl-7,8-dihydro-6H-quinazolin-5-one O-methyl-oxime    20. 2-Amino-7-(2-fluoro-phenyl)-7,8-dihydro-6H-quinazolin-5-one oxime    21. 2-Amino-7-(2-fluoro-phenyl)-7,8-dihydro-6H-quinazolin-5-one O-methyl-oxime    22. 2-Amino-7-p-tolyl-7,8-dihydro-6H-quinazolin-5-one oxime    23. 2-Amino-7-p-tolyl-7,8-dihydro-6H-quinazolin-5-one O-methyl-oxime    24. 2-Amino-7-(2-bromo-phenyl)-7,8-dihydro-6H-quinazolin-5-one oxime    25. 2-Amino-7-(2-bromo-4-fluoro-phenyl)-7,8-dihydro-6H-quinazolin-5-one oxime    26. 2-Amino-7-(2,4-difluoro-phenyl)-7,8-dihydro-6H-quinazolin-5-one oxime    27. 2-Amino-7-(2,6-dimethoxy-phenyl)-7,8-dihydro-6H-quinazolin-5-one oxime    28. 2-Amino-7-benzo[1,3]dioxol-4-yl-7,8-dihydro-6H-quinazolin-5-one oxime    29. 2-Amino-7-(2-morpholin-4-yl-phenyl)-7,8-dihydro-6H-quinazolin-5-one oxime    30. 2-Amino-7-(4-fluoro-phenyl)-4-methyl-7,8-dihydro-6H-quinazolin-5-one oxime    31. 2-Amino-7-(4-fluoro-phenyl)-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-methyl-oxime    32. 2-Amino-7-(4-fluoro-phenyl)-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-(2-dimethylamino-ethyl)-oxime    33. 2-Amino-7-furan-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one oxime    34. 2-Amino-7-furan-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-methyl-oxime    35. 2-Amino-7-furan-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-(2-dimethylamino-ethyl)-oxime    36. 2-Amino-4-methyl-7-phenyl-7,8-dihydro-6H-quinazolin-5-one oxime    37. 2-Amino 1-methyl-7-phenyl-7,8-dihydro-6H-quinazolin-5-one O-methyl-oxime    38. 2-Amino-7-thien-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one oxime    39. 2-Amino-7-thien-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-methyl-oxime    40. 2-Amino-4-methyl-7-p-tolyl-7,8-dihydro-6H-quinazolin-5-one oxime    41. 2-Amino-4-methyl-7-p-tolyl-7,8-dihydro-6H-quinazolin-5-one O-methyl-oxime    42. 2-Amino-7-(2-methoxy-phenyl)-4-methyl-7,8-dihydro-6H-quinazolin-5-one oxime    43. 2-Amino-7-(2-methoxy-phenyl)-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-methyl-oxime    44. 2-Amino-7-(3-fluoro-phenyl)-4-methyl-7,8-dihydro-6H-quinazolin-5-one oxime    45. 2-Amino-7-(3-fluoro-phenyl)-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-methyl-oxime    46. 2-Amino-7-(2-fluoro-phenyl)-4-methyl-7,8-dihydro-6H-quinazolin-5-one oxime    47. 2-Amino-7-(2-fluoro-phenyl)-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-methyl-oxime    48. 2-Amino-7-(2-bromo-phenyl)-4-methyl-7,8-dihydro-6H-quinazolin-5-one oxime    49. 2-Amino-7-(2,6-dimethoxy-phenyl)-4-methyl-7,8-dihydro-6H-quinazolin-5-one oxime    50. 2-Amino-7-benzo[1,3]dioxol-4-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one oxime    51. 2-Amino-7-(4-fluoro-2-pyridin-3-yl-phenyl)-7,8-dihydro-6H-quinazolin-5-one oxime    52. 2-Amino-7-biphenyl-2-yl-7,8-dihydro-6H-quinazolin-5-one oxime    53. 2-Amino-7-biphenyl-2-yl-7,8-dihydro-6H-quinazolin-5-one O-methyl-oxime    54. (2-Amino-7-biphenyl-2-yl-7,8-dihydro-6H-quinazolin-5-ylideneaminooxy)-acetic acid    55. 2-Amino. 7-biphenyl-2-yl-7,8-dihydro-6H-quinazolin-5-one O-(2-morpholin-4-yl-2-oxo-ethyl)-oxime    56. 2-Amino-7-biphenyl-2-yl-7,8-dihydro-6H-quinazolin-5-one O-ethyl-oxime    57. 2-Amino-7-biphenyl-2-yl-7,8-dihydro-6H-quinazolin-5-one O-propyl-oxime    58. 2-Amino-7-biphenyl-2-yl-7,8-dihydro-6H-quinazolin-5-one O-butyl-oxime    59. 4-(2-Amino-7-biphenyl-2-yl-7,8-dihydro-6H-quinazolin-5-ylideneaminooxy)-butyric acid ethyl ester    60. 4-(2-Amino-7-biphenyl-2-yl-7,8-dihydro-6H-quinazolin-5-ylideneaminooxy)-butyric acid    61. 2-Amino-7-biphenyl-2-yl-7,8-dihydro-6H-quinazolin-5-one O-(2-morpholin-4-yl-ethyl)-oxime    62. 2-Amino-7-(2-pyridin-2-yl-phenyl)-7,8-dihydro-6H-quinazolin-5-one oxime    63. 2-Amino-7-(2-pyridin-3-yl-phenyl)-7,8-dihydro-6H-quinazolin-5-one oxime    64. 2-Amino-7-(2-pyridin-4-yl-phenyl)-7,8-dihydro-6H-quinazolin-5-one oxime    65. 2-Amino-7-[4-fluoro-2-(1-methyl-1H-pyrazol-4-yl)-phenyl]-7,8-dihydro-6H-quinazolin-5-one oxime    66. 2-Amino-7-biphenyl-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one oxime    67. 2-Amino-7-biphenyl-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-methyl-oxime    68. 2-Amino-7-biphenyl-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-ethyl-oxime    69. (2-Amino-7-biphenyl-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-ylideneaminooxy)-acetic acid    70. 2-Amino-7-biphenyl-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-(2-morpholin-4-yl-2-oxo-ethyl)-oxime    71. 2-Amino-7-biphenyl-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-propyl-oxime    72. 2-Amino-7-biphenyl-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-butyl-oxime    73. 4-(2-Amino-7-biphenyl-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-ylideneaminooxy)-butyric acid ethyl ester    74. 2-Amino-7-biphenyl-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-(3-morpholin-4-yl-propyl)-oxime    75. 2-Amino-7-biphenyl-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-[3-(4-methyl-piperazin-1-yl)-propyl]-oxime    76. 2-Amino-7-biphenyl-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-[2-(4-methyl-piperazin-1-yl)-ethyl]-oxime    77. 2-Amino-7-biphenyl-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-(3-dimethylamino-propyl)-oxime    78. 2-Amino-7-biphenyl-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-(2-pyrrolidin-1-yl-ethyl)-oxime    79. 2-Amino-7-biphenyl-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-(2-morpholin-4-yl-ethyl)-oxime    80. 2-Amino-7-biphenyl-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-(2-diethylamino-ethyl)-oxime    81. 2-Amino-7-biphenyl-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-(2-dimethylamino-ethyl)-oxime    82. 2-Amino-7-biphenyl-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-(3-piperazin-1-yl-propyl)-oxime    83. 2-Amino-7-biphenyl-2-yl-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-hex-5-ynyl-oxime    84. 2-Amino-4-methyl-7-(2-pyridin-3-yl-phenyl)-7,8-dihydro-6H-quinazolin-5-one oxime    85. 2-Amino-4-methyl-7-(2-pyridin-4-yl-phenyl)-7,8-dihydro-6H-quinazolin-5-one oxime    86. 2-Amino-4-methyl-7-(2-pyridin-2-yl-phenyl)-7,8-dihydro-6H-quinazolin-5-one oxime    87. 2-Amino-7-(5-fluoro-biphenyl-2-yl)-4-methyl-7,8-dihydro-6H-quinazolin-5-one oxime    88. 2-Amino-7-(4-fluoro-biphenyl-2-yl)-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-(3-dimethylamino-propyl)-oxime    89. 2-Amino-7-(4-fluoro-2-pyridin-3-yl-phenyl)-4-methyl-7,8-dihydro-6H-quinazolin-5-one oxime    90. 2-Amino-7-(4-fluoro-2-pyridin-3-yl-phenyl)-4-methyl-7,8-dihydro-6H-quinazolin-5-one O-(3-dimethylamino-propyl)-oxime    91. 2-Amino-4-ethyl-7-(4-fluoro-phenyl)-7,8-dihydro-6H-quinazolin-5-one oxime;and their stereoisomers, tautomers, pharmaceutically acceptable salts, and prodrugs.
Compounds of general formula (I) may be prepared from compounds of general formula (II)
wherein R1, R2, R3, R4, R5, R8 and R9 are as above defined for general formula (I), by reaction with a compound of general formula (III):
wherein R6 is as above defined for general formula (I). Typically, the reaction is conducted in a polar organic solvent such as chloroform or pyridine and it may be necessary to heat the reaction mixture, for example to between about 50 and 80° C.
This method is effective for most R6 groups and in particular can be used for compounds in which R6 is hydrogen, alkyl, alkenyl, alkynyl, (CH2)nC(O)R12, C1-C6 alkylN(R14)2, where n is 1 to 4, R12 and R14 are as defined above, C3-C7 cycloalkyl, C5-C7 cycloalkenyl, aryl, heteroaryl, or C3-C7 heterocyclyl. The choice of solvent will depend upon the nature of the R6 group. When R6 is hydrogen, chloroform may be the preferred solvent, but when R6 is other than hydrogen, pyridine may be a more suitable solvent.
Compounds of general formula (III) are well known and are either readily available or may be prepared by standard methods known to those of skill in the art.
Compounds of general formula (II) in which R1 is hydrogen may be prepared from compounds of general formula (IV):
wherein R2, R3, R4, R5, R8 and R9 are as above defined for general formula (I); by reaction with guanidine hydrochloride in the presence of a base such as sodium carbonate. The reaction is preferably conducted in a hydrophilic solvent such as ethanol and at elevated temperature, typically under reflux.
Compounds of general formula (IV) may be prepared from compounds of general formula (V):
wherein R2, R3, R4, R5, R8 and R9 are as above defined for general formula (I); by reaction with N,N-dimethylformamide dimethylacetal. The reaction is preferably conducted in a hydrophilic solvent such as ethanol and at elevated temperature, typically under reflux.
Compounds of general formula (V) in which R2, R4, R5, R8 and R9 are all hydrogen may be prepared from compounds of general formula (VI):
wherein R3 is as above defined for general formula (I);by reaction with diethylmalonate in the presence of sodium ethoxide, followed by reaction with a strong base such as sodium hydroxide and subsequent acidification with a strong acid such as concentrated hydrochloric acid.
Compounds of formula (VI) may be prepared from compounds of formula (VII):
wherein R3 is as above defined for general formula (I);by reaction with acetone in an aqueous solvent.
Compounds of general formula (VII) are well known in the art and are readily available or may be prepared by standard methods known to those skilled in the art.
Some compounds of general formula (II) are difficult to prepare directly from compounds of general formula (IV). Examples of such compounds are compounds of general formula (II) in which R3 is aryl or heteroaryl substituted with an aryl, heteroaryl, cycloalkyl or heterocyclyl group. These compounds may be prepared from the corresponding compounds of general formula (II) in which R3 is aryl or heteroaryl substituted with bromo by reaction with the appropriate aryl, heteroaryl, cycloalkyl or heterocyclyl boronic acid derivative, as illustrated in Example 4 below.
In an alternative method, compounds of general formula (II) in which R1 is other than hydrogen may be prepared by reacting a compound of general formula (VIII):
wherein R1, R2, R3, R4, R5, R8 and R9 are as above defined in general formula (I); by reaction with guanidine carbonate in a solvent such as ethanol.
Compounds of general formula (VIII) may be prepared from compounds of general formula (IX):
wherein R1, R2, R3, R4, R5, R8 and R9 are as defined in general formula (I); with pyrrolidine.
The reaction may be conducted in a polar organic solvent such as chloroform and typically at a temperature of 15 to 25° C., usually at room temperature.
Compounds of general formula (IX) may be prepared by reaction of a compound of general formula (V) as defined above with a compound of general formula (X):
wherein R1 is as above defined for general formula (I).
Compounds of general formula (X) are well known and are readily available or may be prepared by methods known to those of skill in the art.
Compounds of general formula (I) may also be prepared from other compounds of general formula (I). For example compounds of general formula (I) in which R6 is H or C1-C6 alkyl can be converted into compounds in which R6 is (CH2)n—N(C1-C6 alkyl)2, where n is an integer of 1 to 4, by reaction with a compound of general formula (XI):R6-Cl.HCl  (XI)wherein R6 is (CH2)n—N(C1-C6 alkyl)2, and n is an integer of 1 to 4. The same method may also be used for cyclic amines such as morpholine, in which case the R6 group is an N-morpholino group.
Compounds of general formula (I) where R6 is H can be converted to compounds where R6 is C(O)C1-C6 alkyl by reaction with the appropriate acid anhydride. For example a compound of general formula (I) where R6 is C(O)CH3 may be obtained by reacting the corresponding compound of general formula (I) where R6 is H with acetic anhydride.
Compounds where R6 is (CH2)nCOO(C1-C6 alkyl) can be hydrolysed to give the corresponding carboxylic acid using standard methods of hydrolysis.
Compounds where R6 is (CH2)nC(O)OH can be converted to the corresponding amides by reaction with thionyl chloride to form an acid chloride followed by reaction of the acid chloride with an amine. An example is the preparation of compounds in which R6 is (CH2)nC(O)-morpholino.
As discussed above, compounds of general formula (I) are useful for the treatment of diseases which are mediated by excessive or inappropriate Hsp90 activity such as cancers, viral infection and inflammatory diseases and conditions.
In another aspect, the present invention provides methods for treating viral infection, inflammatory diseases and conditions and proliferative diseases in a human or animal subject in need of such treatment comprising administering to said subject an amount of a compound or composition of formula (I) effective to reduce or prevent such viral infection, inflammatory diseases or conditions or cellular proliferation in the subject.
The invention also provides the compounds of general formula (I) for use in medicine, especially in the treatment of viral infection, inflammatory diseases or conditions and proliferative diseases such as cancer.
In a further aspect there is provided the use of a compound of general formula (I) in the preparation of an agent for the treatment of viral infection, inflammatory diseases or conditions and proliferative diseases such as cancer.
Cancers which may be treated using the compounds of general formula (I) include lung and bronchus; prostate; breast; pancreas; colon and rectum; thyroid; stomach; liver and intrahepatic bile duct; kidney and renal pelvis; urinary bladder; uterine corpus; uterine cervix; ovary; multiple myeloma; esophagus; acute myelogenous leukemia; chronic myelogenous leukemia; lymphocytic leukemia; myeloid leukemia; brain; oral cavity and pharynx; larynx; small intestine; non-Hodgkin lymphoma; melanoma; and villous colon adenoma.
The compound of general formula (I) may be administered in combination with another agent useful in the treatment of cancer and examples of such agents include agents that induce apoptosis; polynucleotides (e.g., ribozymes); polypeptides (e.g., enzymes); drugs; biological mimetics; alkaloids; alkylating agents; antitumor antibiotics; antimetabolites; hormones; platinum compounds; monoclonal antibodies conjugated with anticancer drugs, toxins, and/or radionuclides; biological response modifiers (e.g., interferons and interleukins; adoptive immunotherapy agents; hematopoietic growth factors; agents that induce tumor cell differentiation (e.g., all-trans-retinoic acid); gene therapy reagents; antisense therapy reagents and nucleotides; tumor vaccines; inhibitors of angiogenesis, and the like.
Numerous other examples of chemotherapeutic compounds and anticancer therapies suitable for co-administration with the 2-amino-7,8-dihydro-6H-quinazolin-5-one oxime compounds of the invention are known to those skilled in the art.
In certain embodiments, anticancer agents to be used in combination with compounds of general formula (I) comprise agents that induce or stimulate apoptosis. Agents that induce apoptosis include, but are not limited to, radiation; kinase inhibitors (e.g., Epidermal Growth Factor Receptor [EGFR] kinase inhibitor, Vascular Endothelial Growth Factor Receptor [VEGFR] kinase inhibitor, Fibroblast Growth Factor Receptor [FGFR] kinase inhibitor, Platelet-derived Growth Factor Receptor [PGFR] I kinase inhibitor, and Bcr-Abl kinase inhibitors such as STI-571 [Gleevec or Glivec]); antisense molecules; antibodies [e.g., Herceptin and Rituxan]; anti-estrogens [e.g., raloxifene and tamoxifen]; anti-androgens [e.g., flutamide, bicalutamide, finasteride, amino-glutethamide, ketoconazole, and corticosteroids]; cyclooxygenase 2 (COX-2) inhibitors [e.g., Celecoxib, meloxicam, NS-398, and non-steroidal anti-inflammatory drugs (NSAIDs)]; and cancer chemotherapeutic drugs [e.g., irinotecan (Camptosar), CPT-II, fludarabine (Fludara), dacarbazine (DTIC), dexamethasone, mitoxantrone, Mylotarg, VP-16, cisplatinum, 5-FU, Doxrubicin, Taxotere or Taxol]; cellular signaling molecules; ceramides and cytokines; and staurosparine; and the like.
Preferred anticancer agents for use in combination with compounds of general formula (I) include irinotecan, topotecan, gemcitabine, gefitinib, vatalanib, sunitinib, sorafenib, erlotinib, dexrazoxane, gleevec, herceptin, 5-fluorouracil, leucovorin, carboplatin, cisplatin, taxanes, tezacitabine, cyclophosphamide, vinca alkaloids, imatinib, anthracyclines, rituximab, trastuzumab and topoisomerase I inhibitors.
The compounds of the present invention may also be used to treat other conditions mediated by Hsp90, for example viral conditions such as hepatitis B, hepatitis C and herpes simplex; inflammatory conditions such as rheumatoid arthritis, asthma, multiple sclerosis, type I diabetes, Lupus erythmatosus, psoriasis and inflammatory bowel disease; cystic fibrosis; angiogenesis-related diseases such as diabetic retinopathy, haemangiomas and endometriosis. In addition the compounds may be used to treat brain conditions which may be mediated by Hsp90, for example scrapie or its human equivalent, Creuzfeldt-Jakob disease (CJD), Huntington's disease or Alzheimer's disease or to protect normal cells against chemotherapy-induced toxicity. Another use for the compounds is to resensitise previously resistant fungal strains to antifungal agents such as azoles or echinocandins.
The compound of general formula (I) may be administered in combination with another agent useful in the treatment of inflammation, another anti-viral agent, an anti-fungal agent or an agent useful for treating any of the diseases or conditions listed above.
In yet another aspect of the invention, there is provided a pharmaceutical composition comprising a compound of general formula (I) together with a pharmaceutically acceptable excipient.
The composition may further include one or more additional anti-cancer agent such as those listed above or, alternatively, another anti-inflammatory, anti-viral or anti-fungal agent or an agent useful for treating any of the diseases or conditions listed above.
The pharmaceutical compositions of the present invention may be formulated for administration orally, parenterally, sublingually, by aerosolization or inhalation spray, rectally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. Topical administration may also involve the use of transdermal administration such as transdermal patches or ionophoresis devices. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.
Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-propanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose a fixed oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable nonirritating excipient such as cocoa butter and polyethylene glycols, which are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.
Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, cyclodextrins, and sweetening, flavoring, and perfuming agents.
The compounds of the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, for example, Prescott (ed.), “Methods in Cell Biology,” Volume XIV, Academic Press, New York, 1976, p. 33 et seq.
Compounds of general formula (I) may be administered to a patient in a total daily dose of, for example, from 0.001 to 1000 mg/kg body weight daily and more preferred from 1.0 to 30 mg/kg body weight daily. Dosage unit compositions may contain such amounts of submultiples thereof to make up the daily dose.
The agents to be employed in combination with the compounds of general formula (I) will be used in therapeutic amounts as indicated in the Physicians' Desk Reference (PDR) 47th Edition (1993), which is incorporated herein by reference, or such therapeutically useful amounts as would be known to one of ordinary skill in the art.
The compounds of general formula (I) and the other agents can be administered at the recommended maximum clinical dosage or at lower doses. Dosage levels of the active compounds in the compositions of the invention may be varied so as to obtain a desired therapeutic response depending on the route of administration, severity of the disease and the response of the patient. The combination can be administered as separate compositions or as a single dosage form containing both agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions, which are given at the same time or different times, or the therapeutic agents, can be given as a single composition.
Antiestrogens, such as tamoxifen, inhibit breast cancer growth through induction of cell cycle arrest, that requires the action of the cell cycle inhibitor p27Kip. Recently, it has been shown that activation of the Ras-Raf-MAP Kinase pathway alters the phosphorylation status of p27Kip such that its inhibitory activity in arresting the cell cycle is attenuated, thereby contributing to antiestrogen resistance (Donovan, et al., Biol. Chem. 276:40888, 2001). As reported by Donovan et al., inhibition of MAPK signaling through treatment with MEK inhibitor changed the phosphorylation status of p27 in hormone refactory breast cancer cell lines and in so doing restored hormone sensitivity. Accordingly, in one aspect, the compounds of formula (I) or (II) may be used in the treatment of hormone dependent cancers, such as breast and prostate cancers, to reverse hormone resistance commonly seen in these cancers with conventional anticancer agents.
In hematological cancers, such as chronic myelogenous leukemia (CML), chromosomal translocation is responsible for the constitutively activated BCR-ABL tyrosine kinase. The afflicted patients are responsive to gleevec, a small molecule tyrosine kinase inhibitor, as a result of inhibition of Abl kinase activity. However, many patients with advanced stage disease respond to gleevec initially, but then relapse later due to resistance-conferring mutations in the Abl kinase domain. In vitro studies have demonstrated that BCR-Avl employs the Raf kinase pathway to elicit its effects. In addition, inhibiting more than one kinase in the same pathway provides additional protection against resistance-conferring mutations. Accordingly, in another aspect of the invention, the compounds of formula (I) or (II) are used in combination with at least one additional agent, such as gleevec, in the treatment of hematological cancers, such as chronic myelogenous leukemia (CML), to reverse or prevent resistance to the at least one additional agent.
The present invention will be understood more readily by reference to the following examples.
The following are abbreviations used in the examples:    AcOH: acetic acid    Aq.: aqueous    Boc: tert-butoxycarbonyl    br.s: broad singlet    CHLOROFORM-d: deuterated chloroform    conc.: concentrated    CHCl3: chloroform    CH2(COEt2)2: diethyl malonate    d: doublet    dd: doublet of doublets    DCM: dichloromethane    DMF: N,N-dimethylformamide    DMSO: dimethyl sulfoxide    Et3N: triethylamine    EtOAc: ethyl acetate    EtOH: ethanol    g: gram    GC: gas chromatography    h: hour    H: proton    HCl: hydrochloric acid    HPLC: high performance liquid chromatography    Hz: hertz    IC50 value: the concentration of an inhibitor that causes a 50% reduction in a measured activity    IPA: isopropanol    iPrOH: isopropanol    LC/MS: liquid chromatography/mass spectrometry    m: multiplet    M: molar    MeOH: methanol    MeOD-d4: deuterated methanol    μl: microliter    μM: micromolar    μmol: micromole    mg: milligram    MgSO4: magnesium sulfate    MHz: megahertz    min: minute    ml: milliliter    mm: millimeter    mmol: milli mole    Na2CO3: sodium carbonate    NaOAc: sodium acetate    NaOEt: sodium ethoxide    NaOH: sodium hydroxide    NaOMe: sodium methoxide    Na2SO4: sodium sulphate    NH2OH.HCl: hydroxylamine hydrochloride    nm: nanometer    NMR: nuclear magnetic resonance    ppm: part per million    q: quartet    quin: quintet    s: singlet    sat: saturated    t: triplet    td: triplet of doublets    TFA: trifluoroacetic acid    THF: tetrahydrofuran    TLC: thin layer chromatography    UV: ultraviolet    W: watts
Nomenclature for the compounds disclosed in this application was provided using AutoNom 2000 (Automatic Nomenclature) for ISIS/Base, implementing IUPAC standardized nomenclature. Other compounds, intermediates, and starting materials were named using standard IUPAC nomenclature.
It should be understood that the organic compounds according to the invention may exhibit the phenomenon of tautomerism. As the chemical structures within this specification can only represent one of the possible tautomeric forms, it should be understood that the invention encompasses any tautomeric form of the drawn structure. It is understood that the invention is not limited to the embodiments set forth herein for illustration, but embraces all such forms thereof as come within the scope of the above disclosure.
General Methods
Commercially available reagents and solvents (HPLC grade) were used without further purification.
1H NMR spectra were recorded on a Bruker DRX 500 MHz or a Bruker 400 MHz AV spectrometer or a Bruker DPX 360 or 250 MHz spectrometer in deuterated solvents. Chemical shifts (δ) are in parts per million. Thin-layer chromatography (TLC) analysis was performed with Kieselgel 60 F254 (Merck) plates and visualized using UV light.
Analytical HPLC-MS was performed on Agilent HP1100, systems using reverse phase Atlantis dC18 columns (5 μm, 2.1×50 mm), gradient 5-100% B (A=water/0.1% formic acid, B=acetonitrile/0.1% formic acid) over 3 min, injection volume 3 μl, flow=1.0 ml/min. UV spectra were recorded at 215 nm using a Waters 2487 dual wavelength UV detector. Mass spectra were obtained over the range m/z 150 to 850 at a sampling rate of 2 scans per second using Waters ZMD or analytical HPLC-MS was performed on Agilent HP1100, systems using reverse phase Water Atlantis dC18 columns (3 μm, 2.1×100 mm), gradient 5-100% B (A=water/0.1% formic acid, B=acetonitrile/0.1% formic acid) over 7 min, injection volume 3 μl, flow=0.6 ml/min. UV spectra were recorded at 215 nm using a Waters 2996 photo diode array. Mass spectra were obtained over the range m/z 150 to 850 at a sampling rate of 2 scans per second using Waters ZQ. Data were integrated and reported using OpenLynx and OpenLynx Browser software.
Analytical HPLC-MS was also performed on Shimadzu LCMS-2010EV system (MS, pump, PDA) using reversed phase Water Atlantis dC18 columns (3 μm, 2.1×100 mm), gradient 5-100% B (A=Water/0.1% formic acid, B=acetonitrile/0.1% formic acid) over 7 min, injection volume 3 μl, flow=1.0 ml/min. UV spectra were recorded at 215 nm.
HPLC preparative purification of compounds at low or neutral pH prep was performed by UV directed HPLC performed on Gilson Prep LC modules operated with UniPoint software version 5.1 using reversed phase Waters SunFire Prep C18 OBD columns (5 μm, 19×100 mm), gradient 10-100%, B (A=Water/0.1% TFA, B=acetonitrile/0.1% TFA) over 12 min, injection volume 1.0 ml, flow=26 ml/min. UV spectra were recorded at 215 nm. High pH prep was performed on Gilson Prep LC modules operated with UniPoint software version 5.1 using reversed phase Phenomenex Gemini C18 AXIA columns (5 μm, 100×21.2 mm), gradient 10-100%, B (A=2 mM amm. biocarbonate, buffered to pH 10, B=acetonitrile: 2 mM amm biocarbonate 95:5) over 12 min, injection volume 1.0 ml, flow=26 ml/min. UV spectra were recorded at 215 nm.
Compounds were also purified by HPLC by a mass directed collection trigger which comprises of the following modules operated with Waters FractionLynx V4.0 software:                Waters Micromass Platform LCZ single quadrupole mass spectrometer.        Waters 600 solvent delivery module.        Waters 515 ancillary pumps.        Waters 2487 UV detector.        Gilson 215 autosampler and fraction collectorMass directed HPLC with low pH solvents was performed using reversed phase Waters SunFire Prep C18 OBD columns (5 μm 19×100 mm) gradient 10-100%, B (A-Water/0.1% TFA, B=acetonitrile/0.1% TFA) over 10 min, injection volume 1.0 ml, flow=26 ml/min. UV spectra recorded at 215 nm.        